Polymerase chain reaction (PCR) expression of oligonucleotides is a powerful tool in molecular biology. The PCR product is generally labeled to aid in detection. Various methods of direct and indirect labeling of the PCR product have been developed. Direct labeling involves the incorporation of a labeled monomer during enzymatic synthesis, while indirect labeling refers to post-synthetic introduction of the label.
For example, methods for direct labeling of PCR products are known (see, U.S. Pat. No. 5,242,756 and International Patent Application Publication No. WO 99/65993) In such methods, modified nucleoside triphosphates are incorporated into oligonucleotides during amplification; and conjugation of a xanthene or cyanine label to a nucleoside triphosphate is effected via an amide bond formed with the modified nucleoside triphosphates. Other methods involving conjugation of a biotin or metal chelating label to an oligonucleotide via an amide or hydrazide bond are used to form a desired oligonucleotide-label conjugates (see, U.S. Pat. Nos. 4,707,440 and 4,889,798). Another method for indirect labeling of oligonucleotides involves incorporation of a boronic acid containing nucleoside triphosphate into an oligonucleotide during enzymatic synthesis. Complexation of this boronic acid modified oligonucleotide with a hydroxamic acid derivatized label provides the desired labeled oligonucleotide (see, U.S. Pat. No. 5,876,938).
These methods of oligonucleotide labeling, however, are limited by the lack of stability of the label to the amplification reaction conditions, the inability to selectively and specifically incorporate multiple labels, and the instability of succinimidyl esters. Preparation of activated functionalities, such as succinimidyl esters or maleimides, of labels can be costly, and not even possible in some instances, particularly under PCR conditions.
Thus, due to the limitations of currently available methods as described above, there is a need for efficient methods for labeling of oligonucleotides. Therefore, it is an object herein to provide monomers and methods for labeling of oligonucleotides without the need for post-synthetic modification of the oligonucleotide. It is also an object herein to provide monomers and methods for enzymatic synthesis of modified biopolymers, including oligonucleotides, that can be specifically labeled. A further object herein is to provide the resulting modified oligonucleotides.